Calcium-dependent cell adhesion receptors, or cadherins, have been proposed to mediate synaptic target recognition and synaptogenesis in vertebrate brains. In particular, cadherins are thought to provide the interaction between pre- and post-synaptic partners. Although it is generally accepted that the adhesion activity of cadherins is essential for these functions, the mechanism of action has been in dispute. Cadherins are capable of mediating homotypic and in some cases, heterotypic interaction. It remains unclear how the synaptic specificity can be attributed to the binding specificity of different cadherins and how the cadherin-mediated interactions are regulated. Furthermore, it was recently found that the vertebrate protocadherin CNR (cadherin-related neuronal receptor) and insect N-cadherins can expand their receptor repertoire by alternative splicing. The alternatively spliced cadherin isoforms could largely increase the complexity and specificity of the adhesive interaction in the developing nervous system. We have previously identified N-cadherin for its requirement in R7 target selection. Genomic sequence inspection and RNA transcript analysis reveal that the N-cadherin locus contain three ?exon modules? , each containing two alternatively used exons (exon 7/exon 7p, exon 13/exon 13p and exon 18/exon 18p). Using RT-PCR analysis, we identified 8 N-cadherin isoforms that are generated by the combinatory use of these exon pairs. The predicted 8 N-cadherin isoforms share the same molecular architecture but have divergent amino acid sequences in the extracellular and the transmembrane regions. Semi-quantitative RT-PCR analysis indicates that these exons are utilized differentially in a tissue-specific manner. Exon 7 and exon 13 are predominantly utilized (>85%) in both eye tissue and whole animal. However, exon 18P is the predominant form in the developing eye tissue while in the whole animals, both exons 18 and 18P are found with approximately the same frequency. All N-cadherin isoforms have the same modular structure, including 16 cadherin repeats in the extracellular domain, a transmembrane region, and a cytoplasmic tail. The exon 7 and exon 7p encode for the carboxyl-terminal half of the eighth cadherin repeat, the linker region and the amino-terminal half of the nineth cadherin repeat. And the exons 13 and 13p encode for the eleventh-twelevth cadherin repeats in the similar fashion. Molecular modeling using the known E-cadherin structure as a template reveals that amino acid sequences divergent among the isoforms mapped to two distinct regions. The first region mediates calcium-binding and the second has been implicated in homophilic interaction. This suggests that the different N-cadherin-1 isoform might mediate differential homo- or heterophilic interaction. We express these isoforms in Drosophila S2 cells and test for their ability to induce cell aggregation. Our data indicate that isoforms encoded by exon 7 but not exon 7p are able to mediate both homo- and hetero-philic interaction. These interactions are calcium-dependent as removing calcium in the medium abolishes cell aggregation. Furthermore, the interaction is type-specific since the N-cadherin-1 isoforms can only interact with one another but not with E-cadherin, another Drosophila classic cadherin. Inspecting the genomic sequence close the N-cadherin-1 locus has lead to the discovery of N-cadherin-2, highly homologous to N-cadherin. N-cadherin-2 gene is smaller than the N-cahderin-1 gene and it is likely a result of a gene duplication event. As the genomic sequence analyses fail to resolve the gene structure unambiguously, we performed detailed RNA transcript and RT-PCR analysis on the N-cadherin-2 gene. We uncover 4 types of alternative transcripts at the 5? end of the mRNA and 3 types at the 3? end. Thus, by combinatory use of these variations, the N-cadherin-2 locus can potentially generate 12 different transcripts that are translated into 6 different protein variants. Unlike N-cadherin-1, the alternative splicing of N-cadherin-2 results in protein variants with 3 different molecular architectures. These include one receptor type that is similar to, but shorter than the N-cadherin-1, another with a truncated cytoplasmic region, and the third, a secreted form (Figure 1). All the N-cadherin-2 variants contain 6 cadherin repeats in the extracellular domain. We are investigating the biological function of these variants using cell-based assays. In addition, we will exam the N-cadherin-2 phenotype in R7 target selection. To gain insight into the biological significance of the N-cadherin molecular diversity, we performed comparative genomic analysis in mosquito and another member of the Drosophila family. Drosophila melangaster diverged from the malaria mosquito (Anopheles gambiae) and Drosophila pseudoobscura about 250 and 25 million years ago, respectively. We identified a parallel modular organization in the N-cadherin ortholog of both mosquito and pseudoobscura. In addition, the amino acid sequences specific to the alternative exons are mostly preserved in the three species. The remarkable conservation ranging from the genomic organization to the predicted amino acid sequences is indicative of the functional significany of N-cadherin molecular diversity. Sequence inspection of the N-cadherin neighboring region reveals the existence of 6 N-cadherin-2 genes (designated N-cadherin-2 ~ N-cadherin-7). These N-cadherin-2 genes are more closely related to one another than to the N-cadhein-1 gene. They likely arose from gene duplication events after Drosophila and Anopheles diverged. These N-cadherin-2 paralogs and the N-cadherin ortholog span approximately 2 Mb (or ~0.8%) of the mosquito genome. In addition, the N-cadherin-5 genomic sequence suggests that it might undergo alternative splicing as seen in the N-cadherin.